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Effects of presowing seed treatment of Triticum aestivum L. and Tr. timopheevii Zhuk. wheat with bacteria Bacillussubtilis and B. thuringiensis were studied. Seedling growth, their generation of hydrogen peroxide, and the activities of pathogenesis-related (PR) proteins oxalate oxidase (OxO), peroxidase (PO), proteinase inhibitor (PI), and chitinase upon infection with the causal fungus of bunt Tilletia caries (DC.) Tul. were analyzed. It was found that Bacillus spp. bacteria attenuated the growth suppression of seedlings caused by the bunt infection and alleviated their affection by the pathogen. The found increase in bunt resistance was associated with the boost in the H2O2 level, OxO and PO activities, and transcriptional activities of the chitinase TaChiI gene initiated by Bacillus spp. bacteria. The differences in activation of transcriptional activity of the proteinase inhibitor TaPI gene in soft wheat seedlings under the influence of B. subtilis and B. thuringiensis strains and their mixture were revealed. The results suppose that the wheat resistance to Til. caries may be formed through different paths, which may be related to the nature of the producer organism.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata, to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici, neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology.

Using real-time PCR analysis, the constitutive expression of PR-protein genes encoding thaumatin-like protein (TLP), peroxidase III (TaPero), chitinase (Chitin), glucanase (Glucan), protease inhibitor (PrInh), oxalate oxidase (OxOxid) and lipid transfer protein (Ltp) was studied in collection varieties of winter wheat. It has been shown that plants of varieties with increased resistance to a complex of leaf pathogens have higher constitutive expression levels of Chitin and PrInh genes, and, to a greater extent, of TLP, TaPero and Glucan genes, compared with non-resistant varieties. It is proposed to use constitutive levels of expression of TLP, TaPero and Glucan genes for the selection of winter wheat varietal samples with increased resistance to the complex of leaf diseases.

Over the past few years, three photorespiratory bypasses have been introduced into plants, two of which led to observable increases in photosynthesis and biomass yield. However, most of the experiments were carried out using Arabidopsis under controlled environmental conditions, and the increases were only observed under low-light and short-day conditions. In this study, we designed a new photorespiratory bypass (called GOC bypass), characterized by no reducing equivalents being produced during a complete oxidation of glycolate into CO2 catalyzed by three rice-self-originating enzymes, i.e., glycolate oxidase, oxalate oxidase, and catalase. We successfully established this bypass in rice chloroplasts using a multi-gene assembly and transformation system. Transgenic rice plants carrying GOC bypass (GOC plants) showed significant increases in photosynthesis efficiency, biomass yield, and nitrogen content, as well as several other CO2-enriched phenotypes under both greenhouse and field conditions. Grain yield of GOC plants varied depending on seeding season and was increased significantly in the spring. We further demonstrated that GOC plants had significant advantages under high-light conditions and that the improvements in GOC plants resulted primarily from a photosynthetic CO2-concentrating effect rather than from improved energy balance. Taken together, our results reveal that engineering a newly designed chloroplastic photorespiratory bypass could increase photosynthetic efficiency and yield of rice plants grown in field conditions, particularly under high light.

Depleting the soil weed seedbank is an important integrated weed management strategy that has the potential to foster lasting weed control. Long-term dormancy and decay resistance of weed seeds pose a challenge to weed eradication efforts. Select soil fungi have been shown to cause significant decay of weed seeds. The physical and chemical mechanisms by which seeds in the seedbank defend themselves against pathogens have been well researched. However, very few studies have purposefully investigated the biochemical defence response of seeds. Enzyme-based biochemical seed defences have been detected in dormant and non-dormant seeds, and research supports their function in pathogen defence. This review summarizes current knowledge of the seed defence enzymes polyphenol oxidase, peroxidase, chitinase and oxalate oxidase. The fungal enzymes chitinase, protease and xylanase that function in pathogenesis of seeds in the soil seedbank are also reviewed. Progress in the development and standardization ofin situenzyme analyses fosters our understanding of actual enzyme activity present in soils, while high-throughput microplate techniques promote efficiency and enable greater scope. Application of genomic, proteomic and transcriptomic techniques to glean a deeper and more holistic understanding of the enzymatic interactions of weed seeds and soil fungi in the soil seedbank will support the development of improved integrated weed management strategies.

We investigated the effect of presowing treatment of seeds with salicylic (SA) and jasmonic (JA) acids on the growth of wheat Triticum aestivum L. seedlings, generation of hydrogen peroxide (Н2О2) therein, and transcriptional activity of the genes encoding defense proteins—oxalate oxidase (OxO), peroxidase (PO), and proteinase inhibitor (PI)—upon their inoculation with stinking smut pathogen Tilletia caries (DC.) Tull. SA and JA were found to reduce adverse effect of T. caries on growth of seedlings and the extent of their infection by the pathogen. Improvement of plant resistance to T. caries depended on a stimulatory effect of SA and JA on the formation of Н2О2 in plant tissues and changes in activity of ОхО, PO, and catalase. SA was shown to elevate transcriptional activity of the genes of oxalate oxidase and peroxidase. We detected a considerable stimulatory effect of JA on transcriptional activity of the gene encoding proteinase inhibitor. Revealed differences in the activation of defense proteins pointed to differing mechanisms of SA and JA action on protective potential of wheat plants infected with T. caries.

Germins and germin-like proteins (GLPs) are ubiquitious glycoproteins in the plant kingdom. They play critical roles in both plant development and responses to stresses. Here we report the identification and functional characterization of the GLP gene family in tea plant (Camellia sinensis). By mining the leaf transcriptomes of tea plants, eight genes encoding GLPs were identified, they were designated as CsGLP1-8. Phylogenetic analysis placed the eight CsGLPs into three subfamilies, with CsGLP4 and CsGLP3 in the subfamily I and II, respectively and all the rest in subfamily III. The functions of CsGLPs were further characterized at both biochemical and biological levels. Analysis of recombinant proteins produced by expressing full-length cDNAs of individual CsGLPs in E. coli revealed that all CsGLPs had oxalate oxidase (OxO) or superoxide dismutase (SOD) activities, which are considered as typical biochemical characteristics of GLPs. In addition, expression analysis of individual CsGLP genes during bud sprouting as well as under biotic and abiotic stresses further confirmed their involvements in tea plant development and stress response. In apical buds, CsGLP genes exhibited differential response to phenological changes. CsGLP2 increased more extremely at bud sprouting stage than dormant stage compared with other genes, signifying its potentiality as a bud germination marker for the selection of germplasms in tea breeding. Under drought and H2O2 stresses, the expression levels of CsGLP almost constantly down-regulated, suggesting that bud sprouting was suppressed by water shortage and oxidative stress. For biotic treatment, six genes showed obvious positive response to herbivore-induced damage. Especially, CsGLP1 and CsGLP2 were dramatically induced by geometrid feeding, indicating that these two GLP members may be involved in insect resistance in tea plant. The results of this study will help to better understand the function of GLPs family in tea plants and to further exploit this knowledge for tea breeding.

BACKGROUND: A state of oxalate homeostasis is maintained in patients with healthy kidney function. However, as GFR declines, plasma oxalate (P(ox)) concentrations start to rise. Several groups of researchers have described augmentation of oxalate secretion in the colon in models of CKD, but the oxalate transporters remain unidentified. The oxalate transporter Slc26a6 is a candidate for contributing to the extrarenal clearance of oxalate via the gut in CKD. METHODS: Feeding a diet high in soluble oxalate or weekly injections of aristolochic acid induced CKD in age- and sex-matched wild-type and Slc26a6(−/−) mice. qPCR, immunohistochemistry, and western blot analysis assessed intestinal Slc26a6 expression. An oxalate oxidase assay measured fecal and P(ox) concentrations. RESULTS: Fecal oxalate excretion was enhanced in wild-type mice with CKD. This increase was abrogated in Slc26a6(−/−) mice associated with a significant elevation in plasma oxalate concentration. Slc26a6 mRNA and protein expression were greatly increased in the intestine of mice with CKD. Raising P(ox) without inducing kidney injury did not alter intestinal Slc26a6 expression, suggesting that changes associated with CKD regulate transporter expression rather than elevations in P(ox). CONCLUSIONS: Slc26a6-mediated enteric oxalate secretion is critical in decreasing the body burden of oxalate in murine CKD models. Future studies are needed to address whether similar mechanisms contribute to intestinal oxalate elimination in humans to enhance extrarenal oxalate clearance.

Oxalate oxidase enzyme in monocotyledonous plants, act as a scavenger to breakdown oxalic acid into carbon dioxide and hydrogen peroxide. They perform several important functions in early plant defence responses, including induction of PR protein synthesis and systemic resistance. In the present study oxalic acid, the virulence factor of fungi, was applied to monocotyledonous plants and it was observed that it activates the innate host immune system, along with secretion of oxalate oxidase. The enzymatic activities of other host defence related enzymes like catalase, superoxide dismutase, ascorbic acid oxidase, phenolic content, phenylalanine ammonia lyase were compared between the control and oxalic acid treated (2mM) plants.

Manganese (Mn) is an essential micronutrient with many functional roles in plant metabolism. Manganese acts as an activator and co-factor of hundreds of metalloenzymes in plants. Because of its ability to readily change oxidation state in biological systems, Mn plays and important role in a broad range of enzyme-catalyzed reactions, including redox reactions, phosphorylation, decarboxylation, and hydrolysis. Manganese(II) is the prevalent oxidation state of Mn in plants and exhibits fast ligand exchange kinetics, which means that Mn can often be substituted by other metal ions, such as Mg(II), which has similar ion characteristics and requirements to the ligand environment of the metal binding sites. Knowledge of the molecular mechanisms catalyzed by Mn and regulation of Mn insertion into the active site of Mn-dependent enzymes, in the presence of other metals, is gradually evolving. This review presents an overview of the chemistry and biochemistry of Mn in plants, including an updated list of known Mn-dependent enzymes, together with enzymes where Mn has been shown to exchange with other metal ions. Furthermore, the current knowledge of the structure and functional role of the three most well characterized Mn-containing metalloenzymes in plants; the oxygen evolving complex of photosystem II, Mn superoxide dismutase, and oxalate oxidase is summarized.

Redox reactions affecting the cell wall extensibility proceed in the apoplast of growing cells. The reactions involve dozens of oxidoreductases localized in cell walls (Class I and III heme peroxidases, FAD- and Cu-dependent amine oxidases, oxalate oxidase, ascorbate oxidase, superoxide dismutase, etc.) together with NADPH oxidase and quinone reductase of the plasma membrane. The cell wall extensibility decreases due to peroxidase-catalyzed phenolic cross-links of polymers. Cell growth is proven to be directly dependent on production of reactive oxygen species (ROS) in the apoplast. A special value is attached to hydroxyl radical OH•, which is able to locally cleave polysaccharides and, thus, increase wall extensibility. Generation of OH• results from one-electron reduction of H2O2 and, consequently, is related to the complex of enzymatic and spontaneous reactions of H2O2 turnover in the apoplast. The extensibility also depends on an ascorbate concentration in the apoplast and on a ratio of its oxidized to reduced forms. This dependence is expressed not only in the well-known down-regulation of phenols oxidation but also through pro-oxidant and signal activities. There is only indirect evidence of a role of apoplast-originated redox signaling in the cell growth regulation. In addition to ascorbate, the signaling may supposedly involve ROS, glutathione recycling reactions, numerous redox-sensitive peptides, and proteins localized in the cell wall and the plasma membrane.

We describe an enzymic colourimetric method for determination of oxalate level in urine using arylamine glass-bound sorghum leaf oxalate oxidase and horseradish peroxidase. The method is based on quantification of H2O2 generated from oxidation of urinary oxalate by immobilized oxalate oxidase, by a colour reaction consisting of 4-aminophnazone, phenol and immobilized peroxidase as chromogen. Minimum detection limit of the method was 0.05 mmol/l. Analytical recovery of added oxalate in urine was 96.8± 3.0% (mean ±S.D.). Within and between day coefficient of variation (CV) for urinary oxalate in urine were < 3.5% and

The response of Triticum aestivum L. to infection by Septoria nodorum Berk, a pathogen causing speckled leaf blotch, was studied. The effect of salicylic acid (SA) and jasmonic acid (JA) signal molecules, as well as chitooligosaccharides (COSs) with different acetylation degrees (ADs), on the accumulation of hydrogen peroxide (Н2О2) in wheat leaves and the pathogenesis-related (PR) proteins of oxalate oxidase (AJ556991.1), peroxidase (TC 151917), and proteinase inhibitor (EU293132.1) was investigated. Treatment with the signal molecules inhibited S. nodorum growth and stimulated Н2О2 accumulation, as well as PR gene expression. SA and COS with 65% AD are found to be more efficient in Н2О2 induction and elevation of the transcriptional level of the oxalate oxidase and peroxidase genes. At the same time, JA and COS with 30% AD stimulated transcription of the proteinase inhibitor gene. The results suggest the existence of differential means of defense response induction by signal molecules with more prospects for the regulation of plant immunity.

In this study, we performed the first comparative proteomic analysis of wheat flag leaves and developing grains in response to drought stress. Drought stress caused a significant decrease in several important physiological and biochemical parameters and grain yield traits, particularly those related to photosynthesis and starch biosynthesis. In contrast, some key indicators related to drought stress were significantly increased, including malondialdehyde, soluble sugar, proline, glycine betaine, abscisic acid content, and peroxidase activity. Two-dimensional difference gel electrophoresis (2D-DIGE) identified 87 and 132 differentially accumulated protein (DAP) spots representing 66 and 105 unique proteins following exposure to drought stress in flag leaves and developing grains, respectively. The proteomes of the two organs varied markedly, and most DAPS were related to the oxidative stress response, photosynthesis and energy metabolism, and starch biosynthesis. In particular, DAPs in flag leaves mainly participated in photosynthesis while those in developing grains were primarily involved in carbon metabolism and the drought stress response. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) further validated some key DAPs such as rubisco large subunit (RBSCL), ADP glucose pyrophosphorylase (AGPase), chaperonin 60 subunit alpha (CPN-60 alpha) and oxalate oxidase 2 (OxO 2). The potential functions of the identified DAPs revealed that a complex network synergistically regulates drought resistance during grain development. Our results from proteome perspective provide new insight into the molecular regulatory mechanisms used by different wheat organs to respond to drought stress.

In vitro screening of plant growth-promoting (PGP) traits was carried out using eight Pseudomonas spp., PPR1 to PPR8, isolated from the rhizosphere of Phaseolus vulgaris growing on the Uttarakhand Himalayan range in India. All the isolates were fast growers, positive for catalase, oxidase and urease activities, and utilized lactose and some amino acids. All the isolates were indole acetic acid (IAA) positive, however PPR8 solubilized potassium and zinc along with various other types of inorganic (tricalcium, dicalcium and zinc phosphate) and organic (calcium phytate) phosphates, as well as producing siderophore and ACC deaminase. PPR8 also produced cyanogens, extracellular chitinase, ?-1,3-glucanase, ?-1,4-glucanase and oxalate oxidase. Based on the PGP traits of all isolates, PPR8 was found to be the most potent plant growth-promoting rhizobacteria (PGPR). Further, PPR8 was identified as Pseudomonas sp. PPR8, based on 16S rRNA gene sequencing analysis. Moreover, the PGP activities of PPR8 confirmed it to be a potent biocontrol agent, inhibiting the growth of various plant pathogenic fungi. This study reveals the potential of Pseudomonas sp. PPR8 to be used as a good bioinoculant for growth promotion of common bean and for the protection of important legume crops from various deleterious phytopathogens.

Oxalic acid (OA) is considered as an important pathogenetic factor of some destructive diseases caused by some fungal pathogens such as Sclerotinia sclerotiorum. Oxalate degradation is important for plant health, and plants that contain oxalate oxidase (OXO) enzymes could breakdown oxalate into CO 2 and H 2 O 2 , which subsequently evokes defense responses. However, some species, such as Arabidopsis thaliana, have no oxalate oxidase activity identified to date. The present study aims to develop transgenic Arabidopsis expressing a wheat oxalate oxidase, to test for the response to OA exposure and fungal infection by S. sclerotiorum. The results showed that the transgenic Arabidopsis lines that expressed the wheat OXO exhibited enhanced resistance to OA exposure and S. sclerotiorum infection in the tolerance assays. In the same manner, it could convert OA to CO 2 and H 2 O 2 to a higher extent than the wild-type. Intensive osmotic adjustments were also detected in the transgenic Arabidopsis lines. The higher level of produced H 2 O 2 subsequently induced an elevated activity of antioxidant enzymes including superoxide dismutase (SOD) and peroxidase (POD) in the transgenic Arabidopsis plants. The present study indicated that the expression of a gene encoding wheat OXO could induce intensive osmotic adjustments and hydrogen peroxide related defense response, and subsequently increased tolerance to S. sclerotiorum in transgenic A. thaliana.

Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline “M73,” with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2–3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to wheat. Results are consistent with the hypotheses that (1) dormant seeds are capable of mounting complex responses to pathogens, (2) a diversity of defense enzymes are involved in responses in multiple plant species, and (3) it is possible to identify fungi capable of selective decay of weed seeds without damaging crop seeds, a concept that may be applicable to weed management in the field. While earlier work on seed defenses demonstrated the presence of passive defenses, this work shows that dormant seeds are also quite responsive and capable of activating and releasing defense enzymes in response to a pathogen.

Septoria tritici blotch caused by the fungus Zymoseptoria tritici is one of the most devastating foliar diseases of wheat. Knowledge regarding mechanisms involved in resistance against this disease is required to breed durable resistances. We compared here the expression of defense and pathogenicity determinants in three cultivars in semi-controlled culture conditions. The most susceptible cultivar, Alixan, presented higher necrosis and pycnidia density levels than Altigo, the most resistant one. In Premio, a moderately resistant cultivar, necrosis developed as in Alixan, while pycnidia developed as in Altigo. In non-infectious conditions, genes encoding for PR1 (pr1), glucanase (gluc) and allene oxide synthase (aos) were constitutively expressed at a higher level in both Altigo and Premio than in Alixan, while chitinase2 (chit2), phenylalanine ammonia lyase (pal), peroxidase (pox2) and oxalate oxidase (oxo), were expressed at a higher level in Premio only. Except for aos, all genes were induced in Alixan during the first steps of the infection symptomless phase. Only pox2, oxo, gluc and pal and pal, chs and lox genes were up-regulated at some time points in Altigo and Premio, respectively. Basal cultivar-dependent resistance against Z. tritici could therefore be explained by various genes expression patterns than high expression levels of given genes. During the necrotrophic phase, Z. tritici cell-wall degrading enzyme activity levels were lower in Altigo and Premio than in Alixan, and were associated more with pycnidia than with necrosis. Similar tissue colonization occurred in the three cultivars, suggesting an inhibition of the switch to the necrotrophic lifestyle in Altigo. This article is protected by copyright. All rights reserved.

The influence of chitooligosaccharides (COS) with different degrees of acetylation (DA) on the production of hydrogen peroxide (H2O2) and changes in the level of gene expression of pathogenesis-related (PR) proteins (oxalate oxidase AJ556991.1, peroxidase TC 151917, chitinase AV029935L, proteinase inhibitor EU293132.1) in the roots of the wheat Triticum aestivum L. inoculated with root rot pathogen Bipolaris sorokiniana (Sacc.) Shoenaker was investigated. Differences were detected in plant responses to infection. These differences were due to the pretreatment of COS seeds with differing DA. Our results demonstrated that COS with a DA over 65% more effectively induced accumulation of H2O2 and increased the transcriptional activity of genes of PR-proteins as compared to COS with a DA of 30%. These data suggest an important role for DA in the manifestation of eliciting properties of COS, also in the presence of H2O2.

The effect of 1-methylcyclopropene (1-MCP), which inhibits the reception of ethylene, on the following has been studied: hydrogen peroxide generation, oxalate oxidase activity, peroxidase activity, catalase activity, and lignin accumulation in infected leaves of soft spring wheat (Triticum aestivum L.) cultivars that differ in their resistance to the leaf blotch disease, caused by the hemibiotrophic fungus Septoria nodorum Berk. A decrease in the development of leaf blotch in wheat leaves under the influence of 1-MCP was, on one hand, followed by an inhibition of catalase activity; on the other hand, it was accompanied by an increase in oxalate oxidase and peroxidase activity, as well as an accumulation of H2O2 in tissues and lignin in the infected zone. The role of the ethylene reception system in the defense response of plants to infection with a hemibiotrophic pathogen, that causes leaf blotch disease, is discussed.

Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection of oxygen consumption and carbon dioxide production from the ion currents of their respective mass peaks. 13 C 2 -oxalate was used to allow for accurate detection of 13 CO 2 ( m / z 45) despite the presence of adventitious 12 CO 2 . Steady-state kinetic constants determined by MIMS are comparable to those obtained by a continuous spectrophotometric assay in which H 2 O 2 production is coupled to the horseradish peroxidase catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). Furthermore, we used MIMS to determine that NO inhibits the activity of the CsOxOx with a K I of 0.58 ± 0.06 μM.

Peroxidase and hydrogen peroxide both play important roles in the final stages of the lignification pathway. Peroxidase, in the presence of H2O2 catalyses the oxidation of monolignols to give lignin. In order to examine this process we looked at lignification in transgenic tobacco plants expressing a barley peroxidase gene, HvPrx8, either alone or in combination with a wheat germin gene, g.f 2.8, which encodes oxalate oxidase, thereby providing a source of H2O2. Elevated activity of the antioxidant ascorbate peroxidase was found in plants expressing oxalate peroxidase and was greatly increased by co-expression with the barley peroxidase, although the latter had no effect when expressed alone. An increase was observed in the oxidation of the lignin monomer, syringaldazine in cell lines over-expressing barley peroxidase, while a decrease was observed in double transformants. Plants over-expressing barley peroxidase have elevated levels of lignin deposition compared to that of wild type tobacco plants. Over-expression of the individual enzymes was also shown to enhance heat-induced programmed cell death (PCD) in cell suspension cultures, an effect which was greatly reduced in the double-expressing lines.

Abscisic acid (ABA), H2O2 and nitric oxide (NO) are important signals in gene expression and physiological responses during plant adaptation to environmental stresses. The essential role of NR-derived NO production in ABA and H2O2 induced antioxidant enzymes were studied using transgenic tobacco plants over-expressing Stylosanthes guianensis 9-cis-epoxycartenoid dioxygenase gene (SgNCED1) for elevated ABA level, or over-expressing wheat oxalate oxidase gene (OxO) for elevated H2O2 level in comparison to the wild type. Compared to the wild type, higher levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and nitrate reductase (NR) activities and NO production were observed in all transgenic plants. For investigating the relationship of ABA, H2O2, and NR-produced NO in the induction of antioxidant enzyme activities, an inhibitor of ABA biosynthesis, scavengers of H2O2 and NO, and an inhibitor of NR were used in the experiments. The results indicate that H2O2-induced activities of SOD, CAT, and APX depends on NR-derived NO in OxO transgenic plants, while ABA-induced activities depends on H2O2 and NR-derived NO in SgNCED1 transgenic plants. Compared to unaltered nitrate reductase 2 (NIA2), NIA1 transcript was induced in both types of transgenic plants. It is suggested NR-derived NO is essential for ABA- or H2O2-induced antioxidant enzyme activities.

Summary Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue-specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence-related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue-specific manner for sheath blight resistance.

Oxalate oxidase has potential to act as an oxygen scavenger in active packaging to increase the shelf-life of food and beverages, while simultaneously producing the protective packaging gas carbon ...

Blood and urine oxalate determinations have been limited to the laboratory setting because of complex sample storage and processing methods as well as the need for color spectrophotometry and ion chromatography. We hypothesized that glucometer test strips, impregnated with glucose oxidase and dyes that measure secondary hydrogen peroxide production, could be infused with oxalate oxidase and produce enhanced color changes in the presence of oxalate. By increasing the amount of sodium oxalate in fresh blood, we found that glucometer-measured oxalate increased on a linear scale. In addition, oxalate levels in synthetic urine could be measured using a visual scale, suggesting that strip dwell time or oxalate/oxalate oxidase concentrations could be manipulated to enhance optimal sensitivity. Although further testing is necessary, this simple, first-generation oxometer may eventually allow point of care testing in the home or office, empowering patients with oxalate-based medical conditions and giving healthcare providers real-time oxalate feedback.

Remarkably few enzymes are known to employ a mononuclear manganese ion that undergoes changes in redox state during catalysis. Many questions remain to be answered about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II), the nature of the dioxygen species involved in the catalytic mechanism, and how these enzymes acquire Mn(II) given that many other metal ions in the cell form more stable protein complexes. Here, we summarize current knowledge concerning the structure and mechanism of five mononuclear manganese-dependent enzymes: superoxide dismutase, oxalate oxidase (OxOx), oxalate decarboxylase (OxDC), homoprotocatechuate 3,4-dioxygenase, and lipoxygenase (LOX). Spectroscopic measurements and/or computational studies suggest that Mn(III)/Mn(II) are the catalytically active oxidation states of the metal, and the importance of ‘second-shell’ hydrogen bonding interactions with metal ligands has been demonstrated for a number of examples. The ability of these enzymes to modulate the redox properties of the Mn(III)/Mn(II) couple, thereby allowing them to generate substrate-based radicals, appears essential for accessing diverse chemistries of fundamental importance to organisms in all branches of life.

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of K m for oxalate and V max for the enzyme reaction were 0.015 M and 30 mmol min -1 , respectively.

Glucose has been widely studied as a fuel in biofuel cells because it not only is abundant in nature and in the bloodstream but also demonstrates low volatility, is nontoxic, and is inexpensive. Those qualities coupled with its relatively high energy density qualify glucose as a promising fuel. However, one key to efficient use of this substrate as fuel is the ability to oxidize glucose to CO2 and convert, more efficiently, the chemical energy released upon the redox reactions to electrical power. Most glucose biofuel cells in literature only oxidize glucose to gluconolactone. In this paper, we report the development of a six-enzyme cascade bioanode containing pyrroloquinoline quinone-dependent enzymes extracted from Gluconobacter sp., aldolase from Sulfolobus solfataricus and oxalate oxidase from barley to sequentially oxidize glucose to carbon dioxide through a synthetic minimal metabolic pathway. This bioanode is also capable of performing direct electron transfer to carbon electrode surfaces and elimi...

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species’ (ROS) (superoxide radical and O2·− H2O2) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O2·− and H2O2 compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.

In wheat, little is known about disease resistance inducers and, more specifically, about the biological activities from those derived from endogenous elicitors, such as oligogalacturonides (OGAs). Therefore, we tested the ability of two fractions of OGAs, with polymerization degrees (DPs) of 2–25, to induce resistance to Blumeria graminis f. sp. tritici and defense responses in wheat. One fraction was unacetylated (OGAs–Ac) whereas the second one was 30% chemically acetylated (OGAs+Ac). Infection level was reduced to 57 and 58% relative to controls when OGAs–Ac and OGAs+Ac, respectively, were sprayed 48 h before inoculation. Activities of various defense-related enzymes were then assayed in noninoculated wheat leaves infiltrated with OGAs. Oxalate oxidase, peroxidase, and lipoxygenase were responsive to both OGAs–Ac and OGAs+Ac, which suggests involvement of reactive oxygen species and oxilipins in OGAs-mediated responses in wheat. In inoculated leaves, both fractions induced a similar increase in H2O2 accumulation at the site of fungal penetration. However, only OGAs+Ac led to an increase in papilla-associated fluorescence and to a reduction of formed fungal haustoria. Our work provides the first evidence for elicitation and protection effects of preventive treatments with OGAs in wheat and for new properties of acetylated OGAs.

Glycerol has drawn increasing attention as a possible fuel, because it has many desirable qualities and is abundant due to the fact that it is a byproduct of biodiesel production. Previous research has shown that non-natural enzyme cascades can be used to create a bioanode that can stepwise oxidize glycerol to carbon dioxide. Two of these enzymes are pyrroloquinoline quinone (PQQ) dependant alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) derived from Gluconobacter. The third enzyme, which is responsible for carbon bond cleavage, is oxalate oxidase (OxOx) derived from barley. Previous research has shown that all three enzymes have demonstrated the ability to undergo direct electron transfer to a carbon electrode which allows for a simple and efficient bioanode that completely oxidizes glycerol. In this study, each enzyme was individually immobilized within modified Nafion ® on a glassy carbon rotating disc electrode (GC-RDE) and voltammetric analysis was performed employing different rotation rates in a solution containing each enzyme's respective substrate. This substrate was glycerol for alcohol dehydrogenase, glyceraldehyde for aldehyde dehydrogenase, and mesoxalic acid for oxalate oxidase. From the voltammograms, Levich plots were produced and the solution diffusion coefficient ( D soln ), the membrane diffusion coefficient ( D film ), k CAT , K M , and V MAX were determined.

The effect of Ca2+ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O2−, H2O2, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca2+ in the culture medium of calli. The increase of the concentration of Ca2+ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut (Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three (AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response.

A soluble oxalate oxidase activity has been detected in homogenate of ripened fruits of strawberry (Fragaria ananassa), as confirmed by the stoichiometric relationship between the disappearance of oxalate and utilization of dissolved O2, and generation of H2O2. The enzyme was purified up to apparent homogeneity and had a Mr of 119 kDa with two identical subunits. Km for oxalate was found to be 1.67×10−3 M, and Vmax of 0.741 mmoles ml−1min−1. It retained 76% of its initial activity, when heated at 60°C for 30 min. The enzyme was found to be glycoprotein in nature. The significant increase in the enzyme activity of ripened fruits compared to that in pre-ripened fruit, and decrease in oxalate level (−0.927 correlation with oxalate oxidase) with advancement of ripening indicated the physiological role of enzyme in fruit ripening.

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

This work describes a novel method of urinary oxalate determination based on a catalase model compound MnL(H2O)2(ClO4)2 (L = bis(2-pyridylmethyl)amino)propionic acid). Urinary oxalate is first decomposed by the oxalate oxidase into carbon dioxide and hydrogen peroxide. The later is then disproportionated into water and oxygen by the catalase model compound forming a color compound, which can be spectrophotometrically monitored. The oxalate concentration of the sample is quantified according to the UV-vis absorbance of the formed color compound. This model compound method maintains the specificity and sensitivity of the conventional enzymatic assay. It has several advantages over the traditional enzymatic method, including low cost, simplicity, large linear range, and adjustable assay time. The model compound method showed a very good linearity in the range of 0.002–20 mmol L−1 oxalate, with a detection limit of 2 μmol L−1 oxalate. The mean urinary oxalate determined by this method was 28.6 μg mL−1, standard deviation (SD) was 1.07 μg mL−1, and variation coefficient (CV) is less than 4%. The results are consistent with that acquired from the enzymatic and HPLC methods. The model compound method also showed that the model compounds of the corresponding enzymes can be an alternative to the enzymes, thus the cost of the methods or assays using the enzymes can be greatly decreased.

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H 2 O 2 -generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.

Germination in the soft wheat (Triticum aestivum L.) cultivar Rosella was followed for three days after imbibition by proteomic analysis of the germ tissue. Two-dimensional electrophoresis was performed in triplicate for proteins extracted from embryos dissected from mature grain and from grains germinated for 1, 2, and 3 days. For this period, 63 proteins (in 86 spots) in the germ were identified as decreasing in abundance, 35 proteins (in 60 spots) as increasing in abundance, and 28 proteins (in 39 spots) as exhibiting no significant abundance change. Proteins with significant abundance changes are discussed in relation to physiology; these include proteases, amylases and amylase inhibitors, enzymes in lipid metabolism, proteins related to water stress, cell wall hydrolases, oxalate oxidase, and H+-ATPases. Functions associated with proteins synthesized during the germination period are inconsistent with the embryo of mature grain being fully primed for germination.

Growth inhibition in acid soils due to Al stress affects crop production worldwide. To understand mechanisms in sensitive crops that are affected by Al stress, a proteomic analysis of primary tomato root tissue, grown in Al-amended and non-amended liquid cultures, was performed. DIGE-SDS-MALDI-TOF-TOF analysis of these tissues resulted in the identification of 49 proteins that were differentially accumulated. Dehydroascorbate reductase, glutathione reductase, and catalase enzymes associated with antioxidant activities were induced in Al-treated roots. Induced enzyme proteins associated with detoxification were mitochondrial aldehyde dehydrogenase, catechol oxidase, quinone reductase, and lactoylglutathione lyase. The germin-like (oxalate oxidase) proteins, the malate dehydrogenase, wali7 and heavy-metal associated domain-containing proteins were suppressed. VHA-ATP that encodes for the catalytic subunit A of the vacuolar ATP synthase was induced and two ATPase subunit 1 isoforms were suppressed. Several proteins in the active methyl cycle, including SAMS, quercetin 3-O-methyltransferase and AdoHcyase, were induced by Al stress. Other induced proteins were isovaleryl-CoA dehydrogenase and the GDSL-motif lipase hydrolase family protein. NADPH-dependent flavin reductase and beta-hydroxyacyl-ACP dehydratase were suppressed.

Glycerol has drawn increasing attention as a prospective fuel because it has many desirable qualities and is abundant due to the fact that it is a by-product of biodiesel production. Qualities such as nontoxicity, extremely low vapour pressure, low flammability and high energy density make glycerol very appealing as an energy source. Previous research has shown that partial oxidation of glycerol can occur at enzymatic bioanodes of biofuel cells utilising PQQ-dependent alcohol dehydrogenase (PQQ-ADH) and PQQ-dependent aldehyde dehydrogenase (PQQ-AldDH). In this paper, we describe the use of glycerol for a fuel in an enzymatic biofuel cell that utilizes a three-enzyme cascade on the anode that can accomplish the complete oxidation of glycerol. The bioanode that was developed contained PQQ-ADH, PQQ-AldDH and oxalate oxidase immobilised within a tetrabutylammonium-modified Nafion membrane. Our previous research has shown that glycerol is an effective fuel with the PQQ-ADH and PQQ-AldDH but still was unable to be fully oxidised. With the addition of oxalate oxidase, these glycerol/air biofuel cells have yielded power densities of up to 1.32 mW cm–2, and have the ability to operate at high fuel concentrations. The oxidation products were confirmed with 13C NMR and comprised mainly 13C-labelled carbonate and glycerate.

Barley kernels contain a single oxalate oxidase located in the embryo and aleurone. It is already present in substantial quantities in unmalted grain and increases in activity during germination. It displays a very broad pH optimum: the optimum was at pH 4.0, but the enzyme still displayed more than 50% of its activity at pH 7. Oxalate oxidase is highly resistant to heat. However, its low affinity for oxygen suggests that it probably does not play a major role in the consumption of oxygen in mashing. The decrease in oxalic acid levels late in germination may be a result of oxalate oxidase action. Oxalic acid was not detected in raw barley.

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural.

The effect of salicylic acid (SA) on oxalate oxidase and peroxidase activities and hydrogen peroxide (H2O2) production in leaf cells has been studied in wheat of the susceptible cultivar Zhnitsa infected by Septoria nodorum, a pathogen of wheat leaf blotch. The results show that fungal hyphae spread into interstices between mesophyll cells and that infected tissues contain H2O2. Treatment with SA results in enhanced H2O2 production in mesophyll cells, which is due to activation of oxalate oxidase and peroxidase in the cell wall. It is proposed that the modulating effect of SA on oxidoreductase activities is involved in the induction of protective response to fungal infection in wheat plants.

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.